This application is for renewal of a project concerned with signal transduction during oocyte maturation and fertilization. It addresses the long-standing unanswered question of how luteinizing hormone (LH) causes meiotic resumption in mammalian follicle- enclosed oocytes. Recent findings, which establish that gap junctions between the somatic cells of the follicle close rapidly in response to LH, form the basis for the 3 aims of this project: 1) to investigate if LH-induced gap junction closure in the follicle is caused by TACE protease-mediated activation of the EGF receptor, 2) to investigate whether phosphorylation-mediated closure of Cx43 gap junctions in the follicle cells is required for resumption of meiosis in response to LH, and 3) to investigate the role of gap junction closure in regulating cAMP and cGMP in the oocyte in response to LH. Gap junction permeability will be investigated by microinjection of live follicle-enclosed oocytes with fluorescent tracers, and analysis by 2-photon microscopy and fluorescence recovery after photobleaching. These studies will be complemented by investigations of the phosphorylation of connexin 43 on specific regulatory sites. Concentrations of cAMP and cGMP will be monitored in follicle-enclosed oocytes using newly developed optical probes and confocal microscopy. These methods for live tissue microscopy are a major advance, because they avoid the necessity of disrupting the regulatory environment of the follicle in order to investigate its function. Genetically modified mice will be used, in combination with these microscopic approaches, in order to test the hypothesis that proteolytic processing of EGF receptor ligands and phosphorylation of connexin 43 on identified serines are essential for transduction of signals from the somatic cells of the follicle to the oocyte. The proposed research will advance knowledge of a crucial biological process, and establish a basis for future clinical developments, especially in the treatment of infertility. In vitro oocyte maturation is an emerging component of methods for human in vitro fertilization, and understanding of signaling mechanisms that control oocyte meiosis will facilitate such advances. PUBLIC HEALTH RELEVANCE: The proposed research concerns the mechanisms by which hormonal signals cause mammalian oocytes, stored in the ovary, to develop to the fertilizable stage. This research will advance knowledge of a crucial biological process, and establish a basis for future clinical developments, especially in the treatment of infertility.